The majority of sequencing technologies are based on the natural machinery that living cells use to copy DNA. A nucleic acid processing enzyme called a polymerase reads and then replicates the DNA that constitutes the human genome. DNA is composed of a sequence of the four individual nucleotide bases. The polymerase reads these nucleotide bases one at a time as it copies the DNA, thereby creating a new strand which is complementary to the original. The polymerase will utilise deoxyribose nucleotide triphosphates (DNTPs) or their analogues which are the constituents of DNA. These are initially in solution at high concentrations and are incorporated into the DNA by the action of the polymerase. In order to detect them, the nucleotides bases may be fluorescently labelled so that they are capable of producing a signal. However, there is a problem in that the incorporated label must be distinguished from those present on the DNTPs remaining in solution. The majority of sequencing technologies therefore rely on alteration of the copied bases, together with a degree of purification of the modified DNA, in order to visualise the DNTPs incorporated into nucleic acid strand produced by the polymerase. This process dramatically increases the complexity of a technology and reduces its robustness or reliability, for example as compared with a homogeneous systems that did not depend on any manipulation of this sort.
This process forms the basis of the highly heterogeneous Sanger technique which utilises fluorescent chain terminators and electrophoretic separation of the product. The incorporation of modified bases also forms the bases for the majority of massive parallel approaches, see WO 93/21340 and U.S. Pat. No. 5,552,278. Unfortunately, when many nucleotide bases have been incorporated into the DNA, the total signal becomes overwhelming and it becomes impossible to detect the signal as a new base is added to the growing strand against the background. Some technologies overcome this by removing the label from the DNA chain after reading and before adding a new base. This is a lengthy and demanding process. There are several variants of this approach, but all demand numerous steps to analyse a length of DNA.
It is also important to understand that in such sequencing methods, the signal comes only from the most recently added bases and generally takes advantage of a phenomenon known as fluorescence resonance energy transfer (FRET). In this process, the polymerase is labelled with a fluorescent compound which captures the energy from the fluorescently labelled bases only if they are in very close proximity. Since the closest bases to the polymerase are those that have recently been copied, the sequence can be read during the polymerisation process without further manipulation.
In an example of this technique disclosed in WO 01/163751, the incorporation of labelled bases into an oligonucleotide is measured. There are numerous caveats to this approach since highly labelled oligonucleotides are difficult to produce. More importantly, there is a high degree of collisional fluorescence quenching between labels on the bases that have been added to the copied oligonucleotide. Furthermore, the signal from up to 20 bases will be captured and the signal strength from these labels is erratic due to the helical nature of the product. In a further approach, the gamma-phosphate of nucleotide bases are labelled with an acceptor dye which is cleaved during nucleotide triphosphate incorporation (US Patent Application No: 2003-0064366). The system must be assessed at a high speed due to the natural rate of this reaction.
WO 97/45539 discloses the use of conjugates of a sequence recognizing element (SRE) covalently bound to a reporter group (RG). The reporter group may be an intercalating dye and the sequence recognizing agent is a peptide or a nucleic acid molecule comprising a series of bases or amino acids that is capable of interacting with a target sequence. Where a nucleic acid sequence is used, this application requires that it is sufficiently long to hybridise to a target molecule. Moreover, the only methods disclosed for the synthesis of these conjugates involve the synthesis of the sequence recognizing element with a functional group which is then capable of subsequent reaction to covalently bond to the reporter group.
It remains a problem in the art to develop new sequencing methods, kits and reagents, in particular those which are robust and capable of providing applications such as real time sequencing.